LTB4诱导THP_1巨噬细胞MMP_9表达及在急性冠脉综合征中意义.docVIP

LTB4诱导THP_1巨噬细胞MMP_9表达及在急性冠脉综合征中意义 　　中图分类号:R54 文献标识码 :A 文章编号:1009_816X(2010)04_0259_04 　　 　　DOI:10.3969/j.issn.1009_816X.2010.04.03 Induced MMP_9 Expression by Leukotriene B4(LTB4) in THP_1 Macrophages and Its S ignificance in Patients with Acute Coronary Syndrome(ACS). GE Wei_li, TANG Li_ji ang, JIANG Jian_jun, et al. Department of Cardiology, Tauzhou Hospital, Affiliat ed Hospital of Wenzhou Medical College, Zhejiang 317000 China 　　[Abstract] ObjectiveTo observe the expression of Matrix Metalloproteinase _9(MMP_9) induced by LTB4 in THP_1 macrophages and to explore its significance inpatients with ACS.MethodsDifferentiated THP_1 macrophages was stimulated withLTB4 in different concentration (10_10～10_7mol/L) and for different time (0,6, 12,24 and 48 hours). RT_PCR was used to measure the expression of MMP_9 mRNA andGelatin Zymography was used to evaluate the activity of MMP_9. Serum LTB4, MMP_ 9 and CRP was measured by Enzyme_linked immunosorbent assay(ELISA) in 115 patie nts with ACS and 105 healthy people (control group).Results LTB4(10-10～10-7mo l/L) upregulated MMP_9 mRNA expression in dose_ and time_ dependent manner and a lso enhanced MMP_9 activity. Serum LTB4 was significant higher in patients withacute myocardial infarction (AMI)and unstable angina pectoris(UAP) than that incontrol group (112.77±22.59pg/mL, 86.94±14.81pg/mL and 40.5±8.85pg/mL,respectively. P95%。 　　1.3 RT_PCR:Trizol抽提试剂常规提取细胞总RNA,mRNA逆转录成cDNA。mRNA逆转录反应 :20μL反应体系中含1μg总RNA,20mg/L的oligo(dT)18,1mmol/L dNTPs,20U RNAse inhi bitor,1×reaction buffer,200I_MuLv逆转录酶。于PCR仪上42℃反应60min,72℃灭活10 min,置冰浴。PCR反应:25μL反应体系含1×PCR buffer,20mmol/L MgCl2,200μmol/LdNTPs,2U Taq DNA聚合酶,上下游引物各10μmol/L,β_actin引物10μmol/L逆转录产物 2μL。PCR条件:预变性95℃×5min、变性94℃×45s、退火57℃×45s、延伸70℃×1min,扩增至30个循环,72℃延伸10min,4℃保存。引物序列:MMP_9 sense(5′GAT GCG TGG AG A GTC GAA ATC 3);antisense(5′CAC CAA ACT GGA TGA CGA TG 3),337bp。β_actinsense(5′CGC TGC GCT GGT CGT CGA CA 3);antisense (5′GTC AGG CAC GAT TTC CCG C T 3),619bp。PCR扩增产物电泳(60V×30min)后